This program is devoted to the characterization of metalloenzyme active sites through the use of multinuclear, multifrequency (9; 35; 95 GHz) CW and pulsed electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies. Emphasis is placed on key catalytic intermediates prepared by cryoreduction and rapid freeze-quench ENDOR/ESEEM methods. The studies will focus on enzymes that carry out two, frequently correlated, processes: O2 activation; radical generation; methodology and applications development represent a third component. Particular goals include: 1) Dioxygen-activating iron enzymes: a) Mononuclear nonheme iron centers: cis- Diol dioxygenases; alpha-Keto-acid-dependent dioxygenases; Superoxide reductases; b) Diron centers: Ribonucleotide reductase; c) Heme enzymes: Cytochromes P450; Heme oxygenase; Nitric oxide synthase. 2) Enzymes generating catalytic radicals: The "radical S-adenosylmethionine" superfamily. 3) ENDOR development: Stochastic rapid-passage ENDOR; Extension of ENDOR/ESEEM to Zn enzymes/proteins through study of Co(II)-analogues.